1. Field of the Invention
The present invention relates generally to antimicrobial agents and, more specifically, to indolicidin analogs and methods of using the analogs to reduce or inhibit microbial growth or survival.
2. Background Information
Infections by microorganisms, including bacteria, viruses and fungi, are a major cause of human morbidity and mortality. Although anyone can be a victim of such infection, the sick and elderly are particularly susceptible. For example, hospitalized patients frequently acquire secondary infections due to a combination of their weakened condition and the prevalence of microorganisms in a hospital setting. Such opportunistic infections result in increased suffering of the patient, increased length of hospitalization and, consequently, increased costs to the patient and the health care system. Similarly, the elderly, particularly those living in nursing homes or retirement communities, are susceptible to infections because of their close living arrangement and the impaired responsiveness of their immune systems.
Numerous drugs are available for treating infections by certain microorganisms. In particular, various bacterial infections have been amenable to treatment by antibiotics. However, the prolonged use of antibiotics since their discovery has resulted in the selection of bacteria that are relatively resistant to these drugs. Furthermore, few if any drugs are effective against microorganisms such as viruses. As a result, continuing efforts are being made to identify new and effective agents for treating infections by a variety of microorganisms.
The identification of naturally occurring compounds that act as antimicrobial agents has provided novel and effective drugs. Many organisms protect themselves by producing natural products that are toxic to other organisms. Frogs, for example, produce a class of peptides, magainins, that are highly toxic if ingested, thus providing a defense mechanism for the frog against potential predators. Magainins have been purified and shown to have antimicrobial activity, thus providing a natural product useful for reducing or inhibiting microbial infections.
Natural products useful as antimicrobial agents also have been purified from mammalian organisms, including humans. For example, the defensins are a class of peptides that have been purified from mammalian neutrophils and demonstrated to have antimicrobial activity. Similarly, indolicidin is a peptide that has been isolated from bovine neutrophils and has antimicrobial activity, including activity against viruses, bacteria, fungi and protozoan parasites. Thus, naturally occurring compounds provide a source of drugs that are potentially useful for treating microbial infections.
Upon identifying naturally occurring peptides useful as antimicrobial agents, efforts began to chemically modify the peptides to obtain analogs having improved properties. Such efforts have resulted, for example, in the identification of indolicidin analogs which, when administered to an individual, have increased selectivity against the infecting microorganisms as compared to the individual""s own cells. Thus, the availability of naturally occurring antimicrobial agents has provided new drugs for treating microbial infections and has provided a starting material to identify analogs of the naturally occurring molecule that have desirable characteristics.
Although such natural products and their analogs have provided new agents for treating microbial infections, it is well known that microorganisms can become resistant to drugs. Thus, a need exists to identify agents that effectively reduce or inhibit the growth or survival of microorganisms. The present invention satisfies this need and provides additional advantages.
The present invention relates to analogs of indolicidin, which is a naturally occurring peptide having the amino acid sequence Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-CONH2 (xe2x80x9cIndol 1-13;xe2x80x9d SEQ ID NO: 1). As disclosed herein, indolicidin analogs of the invention have the structure: X1-X2-X3-X4-X5-X6-P-X6-X6-P-X6-X7-X7-X8 wherein X1 is Ile, Leu, Val, Ala, Gly or absent; X2 is Ile, Leu, Val, Ala, Gly or absent; X3 is Pro or absent; X4 is Trp, Phe or absent; X5 is Arg, Lys or absent; X6 is Trp or Phe; X7 is Arg, Lys or absent; and X8 is homoserine, Met, Met-X9-Met or absent, wherein X9 is at least one amino acid; provided that if X1 is present, either X8 is homoserine, Met or Met-X9-Met, wherein X9 is at least one amino acid, or both occurrences of X7 are absent; and further provided that if X2 is absent, X1 is absent; if X3 is absent, X1 and X2 are absent; if X4 is absent, X1, X2 and X3 are absent; and if X5 is absent, X1, X2, X3 and X4 are absent. For example, the invention provides amino terminal truncated indolicidin analogs, including Indol 2-13 (SEQ ID No: 2), Indol 3-13 (SEQ ID NO: 3), Indol 4-13 (SEQ ID NO: 4) and Indol 5-13 (SEQ ID NO: 5), each of which has broad spectrum antimicrobial activity.
The invention also provides indolicidin analogs, wherein at least one of the Trp residues in an amino terminal truncated indolicidin analog is replaced by a Phe residue (xe2x80x9cIndol/Fxe2x80x9d analogs). For example, the invention provides Indol 2-13/F (SEQ ID NO: 11), in which each of the Trp residues in Indol 2-13 (SEQ ID NO: 2) is replaced with a Phe residue.
Amino terminal truncated indolicidin analogs lacking a carboxy terminal amide also are provided. In addition, the invention provides indolicidin analogs comprising, at the carboxy terminus, a homoserine (xe2x80x9cHsexe2x80x9d) residue (xe2x80x9cIndol-Hsexe2x80x9d), such analogs having antimicrobial activity. For example, the invention provides Indol-Hse analogs such as [H2N-]Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-Hse (SEQ ID NO: 24; xe2x80x9cIndol(1-13)-Hsexe2x80x9d), Indol(2-13)-Hse and the like, and similarly provides Indol/F-Hse analogs, including Indol 1-13/F-Hse. Furthermore, the invention provides nucleic acid molecules encoding the indolicidin analogs of the invention or precursors of such analogs.
The ability of an Indol-Hse or Indol/F-Hse analog to exhibit antimicrobial activity provides a means for expressing poly-indolicidin analog polypeptides in vitro or in vivo, wherein each indolicidin analog in the polypeptide is separated by a Met residue or by the sequence Met-X9-Met, such that treatment of the polypeptide with cyanogen bromide results in the production-Indol-Hse or Indol/F-Hse analogs. For example, Met-X9-Met can be Met-Gly-Ser-Glu-Met (SEQ ID NO: 20) or Met-Ala-Arg-Ile-Ala-Met (SEQ ID NO: 21) or the like. Expression, for example, of one mole of Indol 1-13/F-(Met-Gly-Ser-Glu-Met)-Indol 1-13/F-Met (SEQ ID NO: 22), which consists of two unamidated Indol 1-13/F peptides separated by the sequence Met-Gly-Ser-Glu-Met (SEQ ID NO: 20) and including a carboxy terminal Met, followed by treatment with cyanogen bromide results in the production of two moles of Indol 1-13/F-Hse (SEQ ID NO: 23). The expression of such poly-indolicidin analog polypeptides provides a convenient means to produce substantial amounts of indolicidin analog peptides from a host organism, including a microorganism, because the polypeptide form of the analogs does not have substantial antimicrobial activity. In addition, the invention provides fusion polypeptides comprising an indolicidin analog and a peptide of interest, which can be useful, for example, for facilitating purification of an expressed indolicidin analog.
The invention also relates to methods of using an indolicidin analog to reduce or inhibit microbial growth or survival in an environment capable of sustaining microbial growth or survival by contacting the environment with the indolicidin analog. As such, the invention provides methods of reducing or inhibiting microbial growth or survival on a solid surface, for example, surgical instruments, hospital surfaces, and the like. In addition, methods of the invention are useful for reducing or inhibiting microbial growth or survival in an individual, particularly a mammal such as a human. Thus, the invention provides methods of treating an individual suffering from a pathology caused, at least in part, by microbial infection, by administering an indolicidin analog to the individual, thereby reducing the severity of the pathologic condition.